Systemic fungal infections occur most often in individuals with compromised immune systems. The causative agents of such infections are often fungi normally found in the human body or in the environment, but which are rendered noninvasive by a competent immune system. Such fungi are included in the genera Candida, Aspergillus, Cryptococcus, Histoplasma and Coccidioides. The patients most susceptible to these types of infections include those individuals with cancer, diabetes, alcoholism, drug addiction, extensive burns, organ transplants, immune deficiency diseases, and pregnancy. When these patients are on either chemotherapeutic, immunosuppressive and/or anti-bacterial regimens, the likelihood of contracting fungal infections is further increased. See Rippon, in Medical Mycology: The Pathogenic Fungi and the Pathogenic Actinomycetes, Saunders, (1979).
Treatment of systemic fungal infections is primarily limited to two groups of drugs: the polyene antibiotics such as amphotericin B and nystatin, and the imidazoles, such as ketaconazole and miconazole. Structurally, the polyene antibiotics contain three to seven conjugated double bonds. The double bonds are incorporated into a large ring (26 to 44 carbon atoms) lactone. On the opposite side of the macrocycle from the double bonds, the ring is substituted with from 6 to 12 hydroxyl groups mainly in 1,3 relationships but also in 1,2 and 1,4 relationships. Amphotericin B and nystatin possess both an attached aminosugar and a carboxylic acid group. The opposing effects of the lipophilic polyene region and the lipophobic polyol region render polyenes poorly soluble in water.
The preferred treatment of systemic fungal infections is the administration of amphotericin B (Fungizone) because it is the most effective systemic antifungal drug and is associated with the least number of reoccurrences. The polyenes are not absorbed from the gastrointestinal tract following oral administration and have to be administered by I.V. infusion. Amphotericin B and nystatin however both exhibit acute and chronic toxicity to the cells of the patient and thus the doses which may be administered are limited, often preventing complete cure. In fact, nystatin has only been available for inhalation therapy, and oral and topical use for this reason.
The polyene antifungal antibiotics bind readily to sterols present in cell membranes of animal cells, for example cholesterol, and cause disruption of membrane permeability and cell lysis. The toxicity of amphotericin-B in mammals has been found to be greater for certain membrane systems, such as the renal tubules. Clinical use of amphotericin-B has been associated with acute hemolytic crisis and eventual kidney failure at therapeutic dose levels. Medoff, G. and G.S. Kabavashi, N. Eng. J. Med., 303, p. 145-155 (1980); Cohen, J., Lancet 11, p. 532-537 (1982); Graybill, J.R. and P.C. Craven, Drugs, 25, p. 41-62 (1983).
Studies in animals models, and to a limited extent in humans, have shown that amphotericin B incorporated into phospholipid vesicles (liposomes) exhibits decreased host toxicity when used to treat systemic fungal infections. It is expected, based on the fact that nystatin has the same mechanism of action as amphotericin B, that nystatin incorporated into phospholipid vesicles also exhibits decreased host toxicity. Free and liposomal Amphotericin B are approximately equipotent in treating disseminated Candidiasis in mice. (Mehta et al. Biochimica et Biophysica Acta, 770, p. 230-234 (1984)). Therefore, drug doses of liposomal amphotericin B which exceed the maximum tolerated dose for free amphotericin B can be administered which results in a marked improvement in the survival of infected mice. Id. For example, liposome encapsulated amphotericin-B has been used to treat murine systemic fungal infections such as Candidiasis, (Lopez-Berestein et al., J. Infect. Dis. 147, p. 939-945 (1983)), Cryptococcosis (Graybill et al., J. Infect. Dis., 145, p. 748-752 (1982)), and Histoplasmosis (Taylor et al., Am. Rev. Respir. Dis., 125, p. 610- 611 (1982); Adler-Moore et al., Abst. for IXth Congress, Intern. Soc. for Human and Animal Mycol., (1985)), and to treat terminally ill human cancer patients that have not responded to traditional amphotericin-B therapy (Lopez-Berestein et al., Abst. for 23rd. Intersci. Conf. on Antimicrob. Agents and Chemotherapy (1983); Lopez-Berestein et al., Liposomal Amphotericin B for the Treatment of Systemic Fungal Infections in Patients with Cancer: A Preliminary Study, J. Inf. Dis., 151, 704-710 (1985)). Using this type of liposomal drug delivery, the amount of polyene antifungal antibiotic that can be safely administered and the therapeutic index of the drug can be significantly increased.
In most of the studies referenced above, multilamellar vesicles (MLVs) have been used. The size of these vesicles (0.2-5 .mu.) favors the phagocytosis of the drug-containing vesicles by macrophages. When introduced into the bloodstream, fungi are also initially phagocytized by macrophages in the organs of the reticuloendothelial system (RES) such as the liver, lung and spleen. When the fungi invade other tissues, macrophages which are an important part of the immune response to fungal infections, migrate to these sites and become associated with infected sites. Because of the multilamellar composition of the MLVs, encapsulation of a large amount of amphotericin B can be readily achieved. A greater than 90% encapsulation efficiency for MLVs has been reported. Juliano et al., Pharmokinetic and therapeutic consequences of liposomal drug delivery: Fluoro deoxuridine and Amphotericin B as examples, Biology of the Cell, 97, 39-46 (1983). Unfortunately, MLVs are generated as populations of vesicles which are heterogeneous in size, and this makes it very difficult to standardize preparations manufactured in different batches. Such vesicles may be undesirable to administer into mammals because the larger particle sizes (often several microns), and their likely aggregation and fusion to form even larger particles during storage, enhances the possibility of embolism to organs, particularly the lungs, following administration. Taylor et al., Am. Rev. Respir. Dis., 125, p. 610-611 (1982). Finally, unlike small unilamellar particles, MLVs are difficult to sterilize by filtration.
Recently, amphotericin-B has been encapsulated in small unilamellar vesicles (SUVs) (less than 1 micron) as described by Tremblay et al., Antimicrob. Agents Chemother., 26, p. 170-173 (1984) and suspended in saline solution. Higher host survival rates and lower viable colony counts of fungi from the kidneys, liver and spleen as compared with those for unencapsulated drug were observed in mice with disseminated Candidiasis treated with SUV encapsulated amphotericin B. The acute 50% lethal dose (LD.sub.50), a standard measure of acute toxicity, was 11.8 mg/kg as compared to 2.3 mg/kg for unencapsulated drug. Only a 70% encapsulation efficiency was achieved with these SUVs.
The formulations utilized in the Tremblay et al. study also did not include modification of the vesicles to induce preferential uptake by the RES, and as such only a fraction of the SUVs are likely to be taken up by organs containing macrophages. In investigations using egg phosphatidylcholinecholesterol vesicle formulations, similar to those of Tremblay et al., supra, only 40 to 60% of the administered radiolabel used to label the vesicles became associated with major organs of the RES. In earlier work, vesicle formulations with amino-sugar derivatives on their surfaces have been shown to induce chemotaxis and subsequent uptake by polymorphonuclear leukocytes when injected subcutaneously into mice. Mauk et al., Science, 207, p. 309-311 (1980); Mauk et al., Proc. Nat'l. Acad. Sci. (USA), 77, p. 4430-4434 (1980). When SUVs having the 6-aminomannose derivative of cholesterol associated with the membrane are injected intravenously, three-fourths of the radiolabeled vesicles are in the liver and spleen within three hours. Id. Later work by Wu and colleagues confirmed that incorporating an extended amine on a micelle's surface enhances phagocytosis by mouse peritoneal macrophages. Wu et al., Proc. Nat'l Acad. Sci. (USA), 78, p. 2033-2037 (1981). SUVs having an amine modified surface have also been used to label phagocytic cells in vitro, such as leukocytes, to detect sites of infections. U.S. Pat. No. 4,497,791. Until the present invention, however, in situ targeting of SUVs for macrophages of the RES to assist in treating fungal infections has not been achieved.
It is an object of the present invention to provide stable, homogeneous liposomes (SUVs) with encapsulated polyene antifungal antibiotics in commercial quantities, which would remain intact in the bloodstream until entering the macrophage-containing organs of the RES, such as the liver and the spleen. Another object of this invention is to target the SUVs encapsulating amphotericin B to the macrophages which carry the drug directly to sites of fungal infection. This method of delivery could enhance the therapeutic index of the SUV encapsulated drug over unencapsulated drug and lower the acute and chronic toxicity of the drug. Still another object of the invention is to provide a method for using these improved formulations of encapsulated polyene antifungal antibiotics to treat systemic fungal infections.